Journal: Biomolecules

Article Title: Periostin Augments Vascular Smooth Muscle Cell Calcification via β-Catenin Signaling

doi: 10.3390/biom12081157

Figure Lengend Snippet: Treatment with integrin αvβ3 antibody blunts periostin-induced WNT/β-catenin and osteogenic signaling in HAoSMCs. ( A – G ) Relative mRNA expression of WNT7A ( A ), WNT3A ( B ), MMP2 ( C ), PIT1 ( D ), MSX2 ( E ), CBFA1 ( F ) and ALPL ( G ) in HAoSMCs treated with control (CTR) or recombinant human periostin (POSTN) and with mouse IgG as control or integrin αvβ3 antibody (αvβ3Ab). * p < 0.05; ** p < 0.01; *** p < 0.001 (significant difference versus CTR group); † p < 0.05; †† p < 0.01; ††† p < 0.001 (significant difference versus POSTN group).

Article Snippet: The HAoSMCs were treated for 24 h (mRNA expression and protein abundance) or 7 days (ALP activity) with 100 ng/mL recombinant human periostin (stock in PBS, R&D Systems, Abingdon, UK) [ , ], 2 mM β-glycerophosphate (Sigma Aldrich, Vienna, Austria) [ ], 1 nM LGK974 (stock in DMSO, Cayman Chemical, Ann Arbor, MI, USA) [ ], 10 μM XAV939 (stock in DMSO, Cayman Chemical, Ann Arbor, MI, USA) [ ], 10 μM PRI-724 (stock in DMSO, Selleckchem, Planegg, Germany) [ ] and 1 μg/mL anti-integrin αvβ3 antibody (ab78289, Abcam, Cambridge, UK) [ ] or mouse IgG (R&D Systems, Abingdon, UK).

Techniques: Expressing, Control, Recombinant

Journal: PLoS Biology

Article Title: A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and α v β 3 integrin for osteoporosis therapy

doi: 10.1371/journal.pbio.2002979

Figure Lengend Snippet: (A) WT M-CSF (designated M-CSF WT ) constituting a disulfide-bond–linked homodimer. (B) Monospecific M-CSF that can bind α v β 3 integrin (designated M-CSF αvβ3 ) via an RGD motif but not c-FMS because of mutations in positions 9 and 15. (C) Monospecific M-CSF that can bind only c-FMS (designated M-CSF c-FMS ) because of two point mutations that change the RGD motif to RDG, thereby preventing its binding to α v β 3 integrin. (D) Libraries (designated M-CSF RGD ) created by changing two loops in the M-CSF dimerization site to an RGD motif with three random amino acids on each side, thereby enabling binding to α v β 3 integrin. M-CSF, macrophage colony-stimulating factor; RGD, Arginine-Glycine-Aspartic acid; WT, wild type.

Article Snippet: The M-CSF RGD variants, M-CSF c-FMS , and M-CSF αvβ3 proteins were immobilized on the surface of the chip by using the amine coupling reagents sulfo-NHS, 0.1 M, and EDC (1-ethyl-3-[3dimethylaminopropyl]-carbodiimide, Bio-Rad), 0.4 M. To attach the M-CSF RGD, M-CSF c-FMS , and M-CSF αvβ3 variants covalently to the chip, 1 μg of each protein and 3 μg of BSA in 10 mM sodium acetate buffer (pH 4.0) were used to give 1,141, 900, 1,329, 1,272, and 1,337 response units (RUs) for M-CSF c-FMS and M-CSF αvβ3 variants 4.22, 4.24, and 5.6, respectively.

Techniques: Binding Assay

Journal: PLoS Biology

Article Title: A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and α v β 3 integrin for osteoporosis therapy

doi: 10.1371/journal.pbio.2002979

Figure Lengend Snippet: Yeast-displayed mutant pools were tested for binding to (A) 200 nM c-FMS, (B) 500 nM α v β 3 integrin, (C) 250 nM α v β 3 integrin, (D) 100 nM α v β 3 integrin, (E) 20 nM α v β 3 integrin, and (F) 50 nM c-FMS. High target binders were sorted as indicated in each figure with black square- or polygon-shaped gates. M-CSF, macrophage colony-stimulating factor; RGD, Arginine-Glycine-Aspartic acid.

Article Snippet: The M-CSF RGD variants, M-CSF c-FMS , and M-CSF αvβ3 proteins were immobilized on the surface of the chip by using the amine coupling reagents sulfo-NHS, 0.1 M, and EDC (1-ethyl-3-[3dimethylaminopropyl]-carbodiimide, Bio-Rad), 0.4 M. To attach the M-CSF RGD, M-CSF c-FMS , and M-CSF αvβ3 variants covalently to the chip, 1 μg of each protein and 3 μg of BSA in 10 mM sodium acetate buffer (pH 4.0) were used to give 1,141, 900, 1,329, 1,272, and 1,337 response units (RUs) for M-CSF c-FMS and M-CSF αvβ3 variants 4.22, 4.24, and 5.6, respectively.

Techniques: Mutagenesis, Binding Assay

Journal: PLoS Biology

Article Title: A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and α v β 3 integrin for osteoporosis therapy

doi: 10.1371/journal.pbio.2002979

Figure Lengend Snippet: Binding of M-CSF αvβ3 (A and F), M-CSF c-FMS (B and G), 4.22 (C and H), 4.24 (D and I), and 5.6 (E and J) to c-FMS (A-E) and α v β 3 integrin (F-J) at concentrations of 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM. Source data can be found in . M-CSF, macrophage colony-stimulating factor; RUs, response units; SPR, surface plasmon resonance.

Article Snippet: The M-CSF RGD variants, M-CSF c-FMS , and M-CSF αvβ3 proteins were immobilized on the surface of the chip by using the amine coupling reagents sulfo-NHS, 0.1 M, and EDC (1-ethyl-3-[3dimethylaminopropyl]-carbodiimide, Bio-Rad), 0.4 M. To attach the M-CSF RGD, M-CSF c-FMS , and M-CSF αvβ3 variants covalently to the chip, 1 μg of each protein and 3 μg of BSA in 10 mM sodium acetate buffer (pH 4.0) were used to give 1,141, 900, 1,329, 1,272, and 1,337 response units (RUs) for M-CSF c-FMS and M-CSF αvβ3 variants 4.22, 4.24, and 5.6, respectively.

Techniques: Binding Assay, SPR Assay

Journal: PLoS Biology

Article Title: A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and α v β 3 integrin for osteoporosis therapy

doi: 10.1371/journal.pbio.2002979

Figure Lengend Snippet: M-CSF RGD variant 4.22 is shown in pink, c-FMS in cyan, α v in yellow, β 3 in green, and QTSRGDSPS mutant residues in red. M-CSF, macrophage colony-stimulating factor; RGD, Arginine-Glycine-Aspartic acid.

Article Snippet: The M-CSF RGD variants, M-CSF c-FMS , and M-CSF αvβ3 proteins were immobilized on the surface of the chip by using the amine coupling reagents sulfo-NHS, 0.1 M, and EDC (1-ethyl-3-[3dimethylaminopropyl]-carbodiimide, Bio-Rad), 0.4 M. To attach the M-CSF RGD, M-CSF c-FMS , and M-CSF αvβ3 variants covalently to the chip, 1 μg of each protein and 3 μg of BSA in 10 mM sodium acetate buffer (pH 4.0) were used to give 1,141, 900, 1,329, 1,272, and 1,337 response units (RUs) for M-CSF c-FMS and M-CSF αvβ3 variants 4.22, 4.24, and 5.6, respectively.

Techniques: Variant Assay, Mutagenesis

Journal: PLoS Biology

Article Title: A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and α v β 3 integrin for osteoporosis therapy

doi: 10.1371/journal.pbio.2002979

Figure Lengend Snippet: Purified M-CSF RGD variants 4.22 and 5.6 were tested for binding to (A) MDA-MB-231 breast cancer cell line and (B) murine BMMs at different protein concentrations (1 μM, 2.5 μM, and 7.5 μM). The cellular expression levels of c-FMS and α v β 3 integrin are indicated as superscripts. (C) Cell competition binding assay for variant 5.6 in the presence and absence of the two competitors, namely 10 μM cRGD and 5 μM M-CSF WT . Binding of M-CSF c-FMS and M-CSF αvβ3 to the cells is shown for comparison. (D) Tyrosine phosphorylation of c-FMS and (F) serine phosphorylation of Akt in murine BMMs. Different gel runs are separated by a black line. (E) Relative c-FMS and (G) Akt phosphorylation levels of BMMs following incubation with M-CSF c-FMS , M-CSF αvβ3 , and M-CSF RGD variants 4.22, 4.24, and 5.6 in the presence of recombinant M-CSF as a competitor. Chemiluminescence read-outs were quantified by densitometry. Data are means ± SEM of triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001. The aspect ratios of the membranes in panels D and F were changed. Source data and analysis can be found in . BMM, bone-marrow–derived monocyte; cRGD, cyclic RGD; M-CSF, macrophage colony-stimulating factor; MDA-MB-231, MD Anderson metastatic breast 231; RGD, Arginine-Glycine-Aspartic acid.

Article Snippet: The M-CSF RGD variants, M-CSF c-FMS , and M-CSF αvβ3 proteins were immobilized on the surface of the chip by using the amine coupling reagents sulfo-NHS, 0.1 M, and EDC (1-ethyl-3-[3dimethylaminopropyl]-carbodiimide, Bio-Rad), 0.4 M. To attach the M-CSF RGD, M-CSF c-FMS , and M-CSF αvβ3 variants covalently to the chip, 1 μg of each protein and 3 μg of BSA in 10 mM sodium acetate buffer (pH 4.0) were used to give 1,141, 900, 1,329, 1,272, and 1,337 response units (RUs) for M-CSF c-FMS and M-CSF αvβ3 variants 4.22, 4.24, and 5.6, respectively.

Techniques: Purification, Binding Assay, Expressing, Variant Assay, Incubation, Recombinant, Derivative Assay

Journal: PLoS Biology

Article Title: A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and α v β 3 integrin for osteoporosis therapy

doi: 10.1371/journal.pbio.2002979

Figure Lengend Snippet: (A) Murine BMMs were allowed to differentiate into osteoclasts in the presence of M-CSF and RANKL for 72 h. Then, the cells were incubated for an additional 24 h without (positive control) or with inhibitors (5 μM), followed by fixation and staining for F-actin and nuclei. The cells formed solid actin belts (white arrowheads), actin belts with "scattered" podosomes (white arrows), or "amorphous" actin stain distribution (barbed arrowheads). (B) The actin belts’ formation was quantified by normalizing the numbers of solid actin belt to the number of osteoclasts. Pictures are representatives of 35 images acquired from five different wells per sample. Data are means ± SEM of triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001. Source data can be found in . BMM, bone-marrow–derived monocyte; cRGD, cyclic RGD; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of the nuclear factor–kappa-B ligand; RGD, Arginine-Glycine-Aspartic Acid.

Article Snippet: The M-CSF RGD variants, M-CSF c-FMS , and M-CSF αvβ3 proteins were immobilized on the surface of the chip by using the amine coupling reagents sulfo-NHS, 0.1 M, and EDC (1-ethyl-3-[3dimethylaminopropyl]-carbodiimide, Bio-Rad), 0.4 M. To attach the M-CSF RGD, M-CSF c-FMS , and M-CSF αvβ3 variants covalently to the chip, 1 μg of each protein and 3 μg of BSA in 10 mM sodium acetate buffer (pH 4.0) were used to give 1,141, 900, 1,329, 1,272, and 1,337 response units (RUs) for M-CSF c-FMS and M-CSF αvβ3 variants 4.22, 4.24, and 5.6, respectively.

Techniques: Incubation, Positive Control, Staining, Derivative Assay

Journal: PLoS Biology

Article Title: A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and α v β 3 integrin for osteoporosis therapy

doi: 10.1371/journal.pbio.2002979

Figure Lengend Snippet: Murine BMMs were cultured for 96 h in a medium containing recombinant mouse M-CSF (20 ng/ml), RANKL (20 ng/ml), and different concentrations of inhibitors. The same medium without inhibitors was used as a positive control. The medium for the negative control was supplemented with recombinant M-CSF. (A) Cells were fixed and stained for TRAP. (B–D) Cells were examined for: (B) number of osteoclasts, (C) number of nuclei within osteoclasts, (D) total surface area, and (E) total TRAP absorbance were normalized to the positive control. The effect of the inhibitors on markers of osteoclast differentiation was assessed using quantitative PCR for (F) NFATc1 and (G) Oscar mRNA expression. (H) The effect of the inhibitor on pre-osteoclast cell survival was assayed by measuring PI incorporation in osteoclasts cultured for 48 h in the presence of 1 μM of each inhibitor. (I) To test whether there is an unspecific toxic effect of the inhibitors, BMSCs were tested for cell viability in the XTT assay in the presence of three different concentrations of inhibitor (50 nM, 1 μM, and 5 μM). Data are means ± SEM of triplicates. A total of 2,340 frames were analyzed for 1,581 osteoclasts and 5,313 nuclei. * p < 0.05, ** p < 0.01, *** p < 0.001. Source data and analysis can be found in . BMM, bone-marrow–derived monocyte; BMSC, bone-marrow–derived mesenchymal stromal cell; M-CSF, macrophage colony-stimulating factor; PI, propidium iodide; RANKL, receptor activator of the nuclear factor–kappa-B ligand; RGD, Arginine-Glycine-Aspartic acid; TRAP, tartrate-resistant acid phosphatase; XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.

Article Snippet: The M-CSF RGD variants, M-CSF c-FMS , and M-CSF αvβ3 proteins were immobilized on the surface of the chip by using the amine coupling reagents sulfo-NHS, 0.1 M, and EDC (1-ethyl-3-[3dimethylaminopropyl]-carbodiimide, Bio-Rad), 0.4 M. To attach the M-CSF RGD, M-CSF c-FMS , and M-CSF αvβ3 variants covalently to the chip, 1 μg of each protein and 3 μg of BSA in 10 mM sodium acetate buffer (pH 4.0) were used to give 1,141, 900, 1,329, 1,272, and 1,337 response units (RUs) for M-CSF c-FMS and M-CSF αvβ3 variants 4.22, 4.24, and 5.6, respectively.

Techniques: Cell Culture, Recombinant, Positive Control, Negative Control, Staining, Real-time Polymerase Chain Reaction, Expressing, XTT Assay, Derivative Assay

Journal: PLoS Biology

Article Title: A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and α v β 3 integrin for osteoporosis therapy

doi: 10.1371/journal.pbio.2002979

Figure Lengend Snippet: Human CD14 + cells were cultured for 96 h in medium containing recombinant human M-CSF (20 ng/ml), murine RANKL (20 ng/ml), and different concentrations of inhibitors. The same medium without inhibitors was used as a positive control. The αMEM medium for the negative control was supplemented with recombinant M-CSF. (A) Cells were fixed and stained for TRAP. (B-D) Cells were examined for (B) number of mature osteoclasts, (C) number of nuclei within osteoclasts, and (D) total surface area. Results were normalized to the positive control. Data are means ± SEM of triplicates. A total of 1,620 frames were analyzed for 477 osteoclasts and 1,897 nuclei. * p < 0.05, ** p < 0.01, *** p < 0.001. Source data and analysis can be found in . αMEM, alpha Minimum Essential Medium; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of the nuclear factor–kappa-B ligand; RGD, Arginine-Glycine-Aspartic acid; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: The M-CSF RGD variants, M-CSF c-FMS , and M-CSF αvβ3 proteins were immobilized on the surface of the chip by using the amine coupling reagents sulfo-NHS, 0.1 M, and EDC (1-ethyl-3-[3dimethylaminopropyl]-carbodiimide, Bio-Rad), 0.4 M. To attach the M-CSF RGD, M-CSF c-FMS , and M-CSF αvβ3 variants covalently to the chip, 1 μg of each protein and 3 μg of BSA in 10 mM sodium acetate buffer (pH 4.0) were used to give 1,141, 900, 1,329, 1,272, and 1,337 response units (RUs) for M-CSF c-FMS and M-CSF αvβ3 variants 4.22, 4.24, and 5.6, respectively.

Techniques: Cell Culture, Recombinant, Positive Control, Negative Control, Staining

Journal: PLoS Biology

Article Title: A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and α v β 3 integrin for osteoporosis therapy

doi: 10.1371/journal.pbio.2002979

Figure Lengend Snippet: (A) Ex vivo images of 12-weeks-old WT C57BL6 mice organs. The organs were removed and imaged 1.5 h and 3 h after mice were injected s.c. and compared with organs of a mouse that had been injected with unconjugated dye and of a mouse that had not been injected with the proteins (right). To determine whether M-CSF RGD variant 5.6 accumulates in the bones, the epiphysis and diaphysis of the femur of a mouse injected with variant 5.6 were compared with those of the control mice (left). (B) Ten-weeks-old mice were ovariectomized, and starting 2 wk after the surgery, they were injected twice a day with PBS or M-CSF RGD variants 4.22 or 5.6 for 3 d. Thereafter, serum CTX-I levels were determined by ELISA. Data are means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Source data and its analysis can be found in . CTX-I, carboxy-terminal telopeptide of type I collagen; ELISA, enzyme linked immunosorbent assay; M-CSF, macrophage colony-stimulating factor; OVX, ovariectomy-induced bone loss; PBS, phosphate-buffered saline; RGD, Arginine-Glycine-Aspartic acid; s.c., subcutaneously; WT, wild type.

Article Snippet: The M-CSF RGD variants, M-CSF c-FMS , and M-CSF αvβ3 proteins were immobilized on the surface of the chip by using the amine coupling reagents sulfo-NHS, 0.1 M, and EDC (1-ethyl-3-[3dimethylaminopropyl]-carbodiimide, Bio-Rad), 0.4 M. To attach the M-CSF RGD, M-CSF c-FMS , and M-CSF αvβ3 variants covalently to the chip, 1 μg of each protein and 3 μg of BSA in 10 mM sodium acetate buffer (pH 4.0) were used to give 1,141, 900, 1,329, 1,272, and 1,337 response units (RUs) for M-CSF c-FMS and M-CSF αvβ3 variants 4.22, 4.24, and 5.6, respectively.

Techniques: Ex Vivo, Injection, Variant Assay, Enzyme-linked Immunosorbent Assay